Systemically targeted cancer immunotherapy and gene delivery using transmorphic particles

Abstract Immunotherapy is a powerful tool for cancer treatment, but the pleiotropic nature of cytokines and immunological agents strongly limits clinical translation and safety. To address this unmet need, we designed and characterised a systemically targeted cytokine gene delivery system through transmorphic encapsidation of human recombinant adeno‐associated virus DNA using coat proteins from a tumour‐targeted bacteriophage (phage). We show that Transmorphic Phage/AAV (TPA) particles provide superior delivery of transgenes over current phage‐derived vectors through greater diffusion across the extracellular space and improved intracellular trafficking. We used TPA to target the delivery of cytokine‐encoding transgenes for interleukin‐12 (IL12), and novel isoforms of IL15 and tumour necrosis factor alpha (TNFα) for tumour immunotherapy. Our results demonstrate selective and efficient gene delivery and immunotherapy against solid tumours in vivo, without harming healthy organs. Our transmorphic particle system provides a promising modality for safe and effective gene delivery, and cancer immunotherapies through cross‐species complementation of two commonly used viruses.

2 to identify other phage-peptides (2). We and others have published a large body of work proving the tumor selectivity of RGD4C-phage vectors in mice, rats and pet dogs with no gene delivery detected in healthy tissues (3)(4)(5). Recently, we reported that a panel of normal human primary cells from different histological origins do not express or have very low expression of the α v β 3 and α v β 5 integrin receptors of RGD4C (6). Notably, this very low integrin profile did not translate into gene delivery to normal cells by RGD4C/phage vectors. Finally, we also confirmed the tumor selectivity of RGD4C.TPA that we report in this manuscript.

2.
While some progress is reported on minimizing helper phase packaging, the authors still report 2% of particles to be phage. Presumably at very high doses that would be used clinically that level of contamination would be considered a remaining safety issue.
Response: in our study, we reported a contamination of phage in the TPA isolate of 2% or less. This contamination rate is low and increases the novelty of our study, as no phage-derived expression systems utilizing a helper bacteriophage has been able to achieve particle yields while maintaining a contamination rate lower than 10% (7). We understand the reviewer's concern and would like to highlight that in clinical applications, the TPA isolate can be purified to separate the phage contamination using routine methods in the industry, such as ultracentrifugation or fast-protein liquid chromatography, which is currently used to purify bacteriophage preparations in a commercial scale.
We have addressed this concern through clarification in the main text.
3. The authors fail to compellingly provide their rationale for the design considerations of their aav/phage. Why phage? If one is able to make the AAV capsid inert for all tissues, but biodistributing only to tumors, would that achieve the same goal? Why AAV? Do you need the ITRs? What value do they bring? What is the translational relevance of the particular retargeting peptide used, beyond from mouse models? Is less restricted genome size important for the application that the authors pursue? If helper phage genome remains a fairly significant contaminant in either system (albeit reduced in TPA) is the advantage of having smaller particles important in TPA? Do you have stability data to demonstrate this within this system? Response: in our study, we designed and characterised a novel particle, transmorphic phage/AAV (TPA), to address an unmet need in both AAV and bacteriophage as individual vectors due to their native biology. AAV are eukaryotic viruses, which fundamentally lack tissue specificity and are immunogenic, despite being highly efficient at gene expression conferred upon by their ITR sequences. Although it has 3 been shown that serotyping or pseudotyping AAV to distinguished tissue target groups does indeed improve specificity, AAV vectors still cannot safely and systemically target pathology in vivo. In short, the AAV capsid cannot be made selectively inert or highly specific to mammalian tissue due to its inherent biology. Moreover, peptides identified by the phage display technology have been used to generate ligand-targeted vectors to target the cells of interest. For viral vectors, the ideal approach is to genetically engineer new ligands into the capsid proteins of the virus to generate a single agent.
Although this is ideal, this insertion of an exogenous ligand from one structural context into the differing structural context of a capsid protein can ablate the function of the ligand or disrupt viral assembly and function. These "context" problems are fundamental, since an ideal candidate peptide ligand may be identified, by phage display screenings, but cannot be applied because the ligand destroys the vector or the vector destroys the ligand. This translation problem stems in part from the fact that peptides isolated from phage libraries are selected in the protein structural context of the pIII capsid protein of the M13 phage and are then translated into different protein structures of a viral capsid protein (8).
Therefore, we packaged the efficient rAAV transgene cassette (containing ITRs) using the capsid of bacteriophage because of the advantages given by their coat proteins. Specifically, the phage capsid can be modified to be highly specific to tissue targets through insertion mutations (in our case, RGD4C), and are less immunogenic than mammalian viruses as their natural host are prokaryotes; we also reported a large body of literature to show that their repeated administrations, to achieve a therapeutic response, are not a problem. Another important aspect is that bacteriophage-based expression systems will enable industrial-scale production at a fraction of the cost compared to mammalian viruses.
Furthermore, phage vectors have previously been shown to effectively treat soft tissue sarcomas in pet dogs to the point of cure in several subjects, as mentioned in our references. Finally, regarding the role of ITRs, we previously reported that ITR-flanked transgene cassettes provide better gene expression by phage vectors compared to conventional transgenes cassettes, lacking the ITRs, through maintenance of the entire mammalian transgene cassette, better persistence of episomal DNA and formation of concatemers of the transgene cassette(2).
The reviewer brings up an important point concerning particle size. We believe that particle size significantly impacts the efficacy of TPA particles, and that is precisely what we have demonstrated in our study. The particles described is a rAAV genome packaged using bacteriophage coat proteins, which is a key point of novelty as it is truly transmorphic. To be precise, the TPA genome does not contain any phage structural genes, thus differentiating TPA from other hybrid or chimeric phage 4 vectors in existence. The significant reduction in genome size that follows positively impacts its efficacy in gene transduction and cancer immunotherapy, as shown in our in vitro and preclinical data. We investigated the impact of size through our in vitro ECM diffusion assay and Matrigel transwell test, then in eukaryotic cells through a particle internalisation assay 2 and 4 hours post transduction and TPA genome nuclear accumulation. We would like to note that the reduction in genome size also introduces the possibility of packaging larger rAAV transgenes than previously reported in the literature. Because the length of rAAV transgenes is limited by the AAV capsid architecture, using a bacteriophage capsid, which extrudes depending on genome length, will enable transduction of longer or more complex AAV transgenes.
For long term stability, we monitored the TPA titer over 2 years at 4 o C and found that the particles remain fairly stable. Response: as determined by previous studies from our group, we have found no evidence to suggest that the RGD4C mutation is detrimental to replication and encapsulation of TPA particles or bacteriophage vectors. The pIII minor coat protein gene of the bacteriophage is robust and able to tolerate the 9 amino acid mutation on the particular cloning site. In fact, the ability of bacteriophages to tolerate mutations on its pIII gene is hallmark to the development of phage display as a field (awarded the Nobel Prize 2018 in Chemistry). In particular, phage libraries are able to withstand displaying even light chain antibody fragments, demonstrating their ability to tolerate mutations. The question of whether such mutations will affect packaging is a valid one; however we observed high-titre production of TPA particles and do not see that the RGD4C mutation has negatively impacted particle production.
The yields achieved by our production protocol also surpasses those in the literature, including studies on AAVP, indicating novelty and efficiency of the TPA particle expression system.
The reviewer's comments on using genome copies as a measure is a valid criticism that we can perform and benefit from. However, we would like to mention that the infectivity of eukaryotic viruses (i.e. rAAV) cannot be directly compared to prokaryotic viruses or its derivative, as they belong to different native hosts and thus rely on different biological mechanisms of nucleic acid delivery to the nucleus.

5
Another important consideration is economic cost, as the trade-off in infectivity of the TPA (compared to mammalian viruses) may potentially be offset by the cost advantages of using a prokaryotes for its production rather an eukaryotic host cells. 5. Specificity/biodistribution, arguably the defining feature of the innovation in the studies, needs to be demonstrated with more exhaustive molecular analyses and ideally in more relevant pharmacological in vivo models (e.g. NHP?) Response: we are in agreement with the reviewer, however, for the purposes of demonstrating clinical efficacy, we focused on simple but robust methods that demonstrate and confirm straightforward alterations in biodistribution and tumor selectivity. We feel that our experiments provide sufficient mechanistic insight to account for our observations in gene transduction selectivity and immunotherapy in vivo, however, we welcome suggestions on further molecular analyses.

Stylistically
-Two key concepts to the novelty and criticality in the manuscript are barely discussed; technically how is the TPA accomplished; is it really just splitting up the genes in 2 separate plasmids? Also, the targeting and selectivity of RGD4C is assumed. While I understand there is a body of literature on this approach, it is key to the authors argument that this targeting allows for pristine selectivity to be translated to ultimate clinical application. How sure can we be of this?
-Improved articulation of rationale Response: we apologise for the stylistic improvements that the reviewer felt was needed. We have edited the main text to be clear on the design and construction of TPA particles. We would like to point out that the selectivity of the RGD4C is not assumed, but was investigated in the present study and also previously proven in numerous studies from multiple groups that demonstrated that it is an efficacious ligand for binding specifically to tumor cells in vitro and in vivo.

Reviewer #2: "Systemically Targeted Cancer Immunotherapy and Gene Delivery using Transmorphic
Particles" is a manuscript last-authored by Dr. Amin Hajitou, who is an expert in experimental anticancer gene therapy delivery. In this study by Asavarut et al., a novel hybrid vector system based on a bacteriophage (phage) capsid and the DNA of recombinant human adeno-associated virus (AAV) using a tumor-targeted prokaryotic viral capsid has been designed and tested ex vivo and in vivo. A similar 6 vector, termed adeno-associated virus/phage (AAVP), had been previously reported by this group and enabled targeted transgene delivery (2). The AAVP contains the full phage genomic sequence that does not have a therapeutic value and has to be packaged into large particles. Here, the group designed a new system termed Transmorphic Phage/AAV (TPA). It is based on a helper phage which enables packaging of only the essential DNA material in a smaller particle that the authors hypothesized to have a better biodistribution profile. The RGD4C peptide that enables tumor targeting was used as in previous studies for comparison. A hybrid TNFα fused with the IL2 signal peptide and IL15 fused with IgK signal peptide were used as new experimental anti-cancer agents, along with IL12.
This proof of principle study is a significant contribution to the field of targeted cancer therapy. It introduces the new phage-AAV vector as a selective and efficient gene delivery tool. Low TPA particle production, and helper phage contamination were the technical obstacles that the authors encountered and have partly resolved. The use of three different cytokines in various cancer cell models reinforces the conclusions. While the study is important and may have a strong impact on the field, there are a number of technical and conceptual issues that need to be addressed.
Major points: Fig 2C are difficult to tell apart in images provided. It would be better to have filaments at lower density for analysis. Also, the TPA filaments appear to be thicker in Fig 2C: is this the case? If so, is this expected to affect tissue penetration?

Individual phage filaments in
Response: we note that performing the micrograph at lower density will yield a better image. The TPA filaments are not thicker than AAVP, we believe that this is an effect of contrast from the microscope.
We quantified the thickness by measurement during analysis and did not detect any significant differences.

2.
Is it possible that the particle titration method underestimates TPA density relative to AAVP due to worse bacterial infectivity of TPA (which yet might have high cell transducibility)? It would help to confirm titer equality by an alternative method, such as qPCR or immunologically with anti-phage antibodies.
Response: we agree with the reviewer that the study can benefit from using an alternate quantification method for TPA, especially qPCR to determine gene copies. We would like to point out that because the AAVP and TPA both contain pIII minor coat proteins that bear the RGD4C mutation, their bacterial 7 infectivity should be the same as the pIII is the site used by the bacteriophages to bind to the prokaryotic host for entry. If it does indeed affect entry, then it is intrinsically controlled as both AAVP and TPA carries the same mutation on the same protein gene.

For this initial TPA characterization, a side-by-side biodistribution study and evaluation of TPA vs AAVP
targeting is essential in a mouse tumor model. Comparative immunohistochemistry with anti-phage antibodies needs to be performed. GFP or Luc tumor delivery with TPA vs AAVP also needs to be compared.
Response: the main objective of the study was to develop a novel phage vector for application in cancer immunotherapy. We included AAVP in the initial experiments as control for size to show that the reduced size of M13 phage-derived vectors does matter for gene delivery to mammalian cells as this is a growing hypothesis in the field.
We understand the reviewer's point, and that's why we report here the superiority of TPA to AAVP in various in vitro experiments. To comply with the reviewer, we can also carry out in vivo comparison in tumor-bearing mice to confirm the superiority of TPA to AAVP, for gene delivery to tumors, by using particles carrying either Luc, TNF, IL15 or IL12 transgenes. Response: we have generated data in vitro to show that RGD4C.TPA particles provide better delivery of our newly designed TNF compared to RGD4C.AAVP. As mentioned in the above point-3 raised by this reviewer, we also can carry in vivo studies to analyse delivery of TNF to tumors in preclinical models upon systemic administration. Response: we can provide brightfield cell images. The reviewer's observation that GFP expression by RGD4C.AAVP.GFP was too low is an important point. Indeed, the transduction efficiency of RGD4C.AAVP particle can be highly variable for the same cell type and reach sometime low levels around 0.3%. We still don't know the factors behind this, but the TPA always performed better than AAVP for gene delivery, in all experiments where the two particles were compared side-by-side.
-While the prediction that smaller particles should have better tissue permeability are reasonable and confirmed with the Matrigel experiments (Fig 3), it is not clear why TPA transduces so much better in cell culture experiments ( Fig 2F) were there is no ECM hinderance: is there an explanation? Could it be due to some TPA phage not accounted for by bacterial titration?
Response: as we stated in the manuscript improved diffusion through the ECM is one mechanism through which TPA can transduce cells better than AAVP. However, we also report that TAP particles have better cell entry and better nuclear accumulation which are important steps in gene delivery efficacy.
- Fig 3E: why did AAVP remain predominantly in the cytoplasm? What explains better nuclear localization of TPA DNA?
Response: important point. We believe that the TPA contains and delivers the AAV genome only which has evolved optimised mechanisms for intracellular trafficking in mammalian cells. While AAVP delivers both AAV but also a large bacteriophage genome that has no optimised strategies for intracellular trafficking in mammalian cells. there is no integration, and that the AAVP genome remains extrachromosomal. Regarding the long-term transgene expression in tumors in vivo, this can vary depending on the tumor type and how fast the tumors become necrotic(2, 3). We will be happy to include a discussion on this in the manuscript.
-It is mentioned once that RGD4C, the peptide used for phage tumor delivery, binds to αv integrins.
Because αv integrins are also expressed outside tumors, some more discussion of the results and future perspectives are warranted.
Response: the tumor selectivity of RGD4C peptide was also raised in point-1 of reviewer#1 (please see our response).
The RGD4C ligand binds mainly to  v  3 , but also to a lesser level to the  v  5 heterodimer. Various integrin heterodimers can be found in wide variety of human cells; however, both  v  3 and  v  5 are highly restricted and typically overexpressed on cancer cells and tumor vasculature (1,9). In Human biopsies, the  v  3 integrin is widely expressed on blood vessels of human tumor biopsy samples but not on vessels of biopsies from normal tissues; the distribution of  v  3 in human is highly restricted, with expression on activated endothelium, activated vascular smooth muscle and tumors. Besides this, the  v  3 has been shown to have relatively limited cellular distribution in humans quiescent tissues; apart from its expression at high levels in the inflamed synovial tissues of rheumatoid arthritis patients (10,11),  v  3 is absent or minimally, or barely detectable on endothelial cells(12), some B-cells, platelets, monocytes, intestinal cells, and smooth muscle cells, as well as a small percentage of activated leukocytes, macrophages, and osteoclasts. Integrin  v  5 is often found in the same pathological contexts as α v β 3 , but can also be found in fibroblasts. Importantly, we previously reported that there is no expression or minimal expression of these integrins in human normal cells, but this low level of αv integrins does not permit any gene expression by RGD4C.phage vectors(6).

-In mouse experiments, do primers used for RT-qPCR detect only the delivered cytokine gene or also the endogenous cytokine?
Response: no cytokine mRNA signal was detected in control untreated cells.

FASEB J. 2021). In that study, an endosomal escape peptide, H5W, was displayed on pVIII coat protein to enhance gene delivery. It is not clear why the authors chose to not do it for TPA and discussion along these lines would be welcome.
Response: we have data showing that display of the endosomal escape peptide, H5W, on a major pVIII coat protein of the TPA particles further enhances gene delivery efficacy. We will be happy to include these data.

-Results contain a lot of technical details on vector construction, characterization and optimization that appear to be better suited for Methods and/or Supplemental Materials. Also, some parts of Results, such as cancer type descriptions and cytokine MOA seem to belong better in Introduction or Discussion.
Text needs to be proof-read. There are typos and language imperfections. It is advised that the manuscript is edited for clarity. For example, the meaning of the sentence "Furthermore, 90% of patients die from metastatic cancer, which relies on the systemic delivery (6)." Is not clear. Reference call out format is not internally consistent.
Response: we have complied and revised the text to improve the manuscript and will continue to do so.

-TNFα has both anti and pro-cancer effects. For example, it has been reported that TNFα interferes with immune checkpoint blockade approaches to cancer treatment (e.g. Bertrand et al 2017) and can increase cancer cell aggressiveness (W. Liu et al Scientific Reports 2020). This, along with issues related to
potential adverse effects of IL15 and IL12, need to be discussed.
Response: yes, it is correct that TNFα can have both anti and pro-cancer effects but this depends on the levels of its release and production within the tumors and whether its production is selectively localised or not. We and other groups have reported that selective delivery of TNF by RGD4C.phages results in anti-tumor effects in rodents and large animals e.g., pet dogs with natural cancers (4,(13)(14)(15)(16)(17).
26th Jan 2022 1st Editorial Decision 26th Jan 2022 Dear Amin, Thank you for submitting your work to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you. I had originally secured an advisor who promised a report by the end of 2021, but never got back to us despite several chasers. I therefore contacted a second advisor, who has now provided a feedback. This advisor stated: "I have now had a look at the manuscript by Asavurat et al, the reviewers comments and the response to these. I think overall the data is convincing, well presented and supports the conclusions drawn. In particular, the in vivo efficacy of the TPA approach across different tumour models in both immunocompetent and immunosuppressed mice is impressive. In terms of the initial reviewers' comments, which are very thorough and overall positive, in my view these have been thoughtfully and sufficiently addressed. The major point to raise concerning the rebuttal is that, in response to reviewer 2, points 3 and 4, the authors respond that they can compare TPA and AAVP for gene delivery in vivo, although they have not actually done this. However, I think this is acceptable, since they have clearly demonstrated this superiority in vitro. I personally would not request further in vivo experiments for this manuscript. Hence, in summary, I think this is a high-quality paper, for which the initial reviewers' concerns have been sufficiently addressed. I would therefore be happy to now recommend it to be accepted for publication." Based on this advisor's input and after discussion within the team, I am pleased to inform you that we will be able to accept your manuscript, once editorial revisions will be performed. We require:

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2/ Thank you for providing The paper explained. I added minor modifications, mostly to shorten the text, please amend as you see fit: Medical Issue Since the 1980s, antitumor cytokines have been used as cancer immunotherapy. Yet a fundamental problem remains the control over immune activating cytokines at the target site, which can have fatal effects on the host. Cytokine-encoding genes have thus been developed to express cytokines in cancer cells only; however, gene delivery is hindered by the lack of tumor-selective vectors and issues linked to repeated administrations.

Results
We established a unique prokaryotic viral-based approach of intravenous gene delivery to specifically target tumors by using the filamentous M13 bacteriophage that infects bacteria only. In this vector, the M13 phage capsid was engineered to target cancer and deliver therapeutic transgene expression cassettes carrying genes encoding interleukin IL12, IL15 and tumor necrosis factor alpha. These phage-derived particles proved to be an efficient platform for safe and selective systemic delivery of cytokines to solid tumors, while avoiding healthy tissues in preclinical models of human and murine tumors. Moreover, administration of particles in immunocompetent animals could be repeated and resulted in tumor eradication and complete response in more than 50% of the mice.

Clinical Impact
The newly developed phage-derived particles can be applied for selective and efficient cytokine therapy. These findings are important since targeted cytokine delivery has been a major barrier for clinical translation. Given that cytokines have already been tested in cancer patients, and that phage safety in human is increasingly established, the clinical efficacy of this targeted cytokine therapy to treat solid tumors is promising. Moreover, the treatment is administered through the systemic route, and thus could be applied both for localised and metastatic disease.
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